Method for genetic transformation of woody trees

ABSTRACT

The invention refers to a method to transform and obtain plants of woody trees, particularly Eucalyptus spp., transformed by the introduction of exogenous genes of interest into their cells. The method makes use of sterilized seeds, which are co-cultivated with Agrobacterium cells containing a transference plasmid (tDNA), and any gene of interest. The method used for the transformation employs sonication to increase the efficiency of bacteria penetration within teguments and seed embryo tissues.

FIELD OF THE INVENTION

[0001] The invention refers to a method to transform and obtain woodytree plants, particularly Eucalyptus spp., transformed by theintroduction of exogenous genes of interest into their cells. It alsorefers to the use of such transformed plants.

[0002] The method makes use of sterilized seeds, which are co-cultivatedwith Agrobacterium, cells containing a transference plasmid (tDNA) andany gene of interest. The technique used for the transformation utilizessonication to increase the efficiency of bacteria penetration withinteguments and embrionary tissues of the seeds.

[0003] This method for the genetic transformation of woody trees,particularly Eucalyptus spp., belongs to the chemical technical field inthe area of mutation or genetic engineering.

BACKGROUND OF THE INVENTION

[0004] As known, eucalyptus is widely planted in Asia, South America andsome parts of Europe, especially in the Mediterranean region. This kindoffers a range of advantages over other forest species for use in thecellulose industry, such as quick growing, regeneration from multiplesprouting after harvesting and capacity to grow in poor soils. Variousresearch groups in many countries, including public and privateinstitutions have attempted genetic transformation of eucalyptus. Fewreports have however shown stable gene transference for differenteucalyptus species.

[0005] Serrano et al., in their article “Genetic transformation ofEucalyptus globulus through biollistics: complementary development ofprocedures for organogenesis form zygotic embryos and stabletransformation of corresponding proliferation tissue”, J. Exp. Botany 47(295), 1996, 285-290, discloses a particle bombing (bioballistic) methodto introduce DNA molecules containing gene uidA coding theβ-glucuronidase (GUS) protein into zygotic embryos of Eucalyptusglobulus. After two months of bombing, callus were obtained whichpresented positive GUS reaction. Although tDNA integration was shown byhybridization through the Southern Blot method, plants were notregenerated.

[0006] Moralejo et al., “Generation of transgenic Eucalyptus globulusplantlets through Agrobacterium tumefaciens mediated transformation”, J.Plant Physiol., 25:297-212, 1998, obtained transgenic E. globulus plantsby making use of the gene transference method via Agrobacteriumtumefaciens, infecting plantlets which had been injured withmicro-particles bombing before the co-culture. However, the efficiencyin obtaining transgenic plants was very low, around 1%.

[0007] Mullins et al., “Regeneration and transformation of Eucalyptuscamaldulensis”, Plant Cell Reports, 16, 1997, 787-791, obtained plantregeneration from sprouts regenerated from foliar explants andtransformation of a clone of E. camaldulensis, by making use of thetransformation system as mediated by Agrobacterium tumefaciens.

[0008] Ho et al., “Agrobacterium tumefaciens-mediated transformation ofEucalyptus camaldulensis and production of transgenic plants”, PlantCell Rep., 17, 1998, 675-680, also described the transformation andregeneration of E. camaldulensis, by making use of the Agrobacteriumsystem to transform hypocotyls. In all these examples, lowtransformation efficiency was the main limiting factor to thelarge-scale use of such methods to introduce exogenous genes ineucalyptus.

[0009] Various methods have been used to optimize the efficiency ofeucalyptus transformation by making use of the Agrobacterium system.Recently, a transformation system was developed using ultrasound andsonication to increase the efficiency of Agrobacterium penetrationwithin target tissues for transformation. The method called SAAT(“Sonication Assisted Agrobacterium Transformation”) was used toincrease the efficiency of gene transference in soy, peas, wheat andcorn (Trick & Finer, “SAAT-sonication assisted Agrobacterium mediatedtransformation”, Transg. Res., 6:329-336, 1997, “Sonicated assistedAgrobacterium mediated transformation of soybean [Glycine max (L.)Merrill] embryogenic suspension culture tissue”, Plant Cell Rep.,17:482-488, 1998, Santarém et al., “Sonication assisted Agrobacteriummediated transformation of soybean immature cotyledons: optimization oftransient expression”, Plant Cell Rep., 17:752-759, 1998).

[0010] The use of ultrasound for plant tissues induces acousticcavitation, generating microscopic injuries, which are channels to favorthe internal exposure of tissues to Agrobacterium (Joersbo & Brunstedt,“Sonication: A new method for gene transfer to plants”, Physiol. Plant,85:230-234, 1992), thus increasing tDNA transitory expression levels.For eucalyptus, most tissues used for the genetic transformation arecotyledons, hypocotyls, immature embryos and callus obtained fromdifferent plant tissues.

[0011] A bioballistic or biollistic method is also known, consisting infiring micro-projectiles of gold or platinum containing precipitated DNAon their surfaces. The projectile goes through the cellularwall/membrane carrying DNA with it. To launch projectiles, equipmentsimilar to a pressure rifle is used, in which pressure is produced by aninert gas. Such procedure is used for the genetic transformation ofplants and animals.

[0012] Still on this issue, for chemical analysis procedures, ultrasonicwave generator systems are employed to prepare samples, be it inprocesses to extract chemical species or be it to dissolve solidsamples. The base for ultrasonic application to such processes isrelated to the shock waves resulting from the application of an acousticfield over a material means. Such waves increase the interaction betweenthe solvent and the surface of the solids, increasing the concentrationof species present in the material being investigated in the solution.The vibration caused by ultrasounds minimizes the concentration gradientin the surroundings of the surface of the solid present in the exposedmedium and makes possible the transport of salts and oxides from thesurface of the solid to the solution. When ultrasound probes areemployed, however, the association of magnetic agitators to the systemshould not be disregarded, as only a small zone of the solution, inwhich the probe is immersed, will be submitted to the high intensityproduced by such processors and vigorous stirring provides the samelevel of interaction for the whole exposed solution.

[0013] In the sonication method, rupture of cells is obtained, thusallowing for gene introduction for the final genetic transformation. Theuse of sonication aims at increasing the transformation efficiency,since it causes injuries to the tissues, which then release phenolcompounds, increasing the attraction of bacteria to those regions andfacilitating gene transference.

[0014] The previously used bioballistic method for Eucalyptus did notencompass regeneration with β-glucuronidase (GUS) to the recovery of thefinal plant.

[0015] A method to use Agrobacterium with final regeneration into E.camaldulensis under low efficiency has also been reported.

[0016] Patent application WO 9625504-dated Aug. 22, 1996 describes aprocess to obtain transgenic plants by means of incorporation of astable DNA sequence of interest. The process comprises cells or tissuesof Eucalyptus with Agrobacterium being the mediator for the DNA sequencetransference, inducing the formation of callus with phenyl ureaderivatives in the presence of geneticin (G-418). The process is usefulto transform clone material directly or indirectly derived from a matureEucalyptus tree.

BRIEF DESCRIPTION OF THE INVENTION

[0017] Due to the limitations of all known methods so far, forbiddingtheir use in large scale for the introduction of exogenous genes ineucalyptus, the present method has been studied, developed and arrivedat, for the genetic transformation of woody trees, particularly,Eucalyptus spp. Said method, a fully new technique within this field ofindustrial activity, unexpectedly facilitates the genetic transformationof woody trees, particularly, Eucalyptus spp.

[0018] With this purpose, the method for genetic transformation of woodytrees, particularly, Eucalyptus spp. at issue has as a main andundisclosed technical characteristic the genetic transformation viaseeds of woody trees, particularly, Eucalyptus spp., thus offering thepossibility to increase substantially the efficiency of the process.

DESCRIPTION OF FIGURES

[0019] These and other objects of this invention will be apparent to theone skilled in the art from the attached schematic figures, where:

[0020]FIG. 1 is a view of the expression of β-glucuronidase (GUS) genein 2-day germination seeds (A to C) and 15-day plantlets (D to F);

[0021]FIG. 2 shows the regeneration of explants from hypocotyls,cotyledons and leaves, as obtained by the use of phenyl urea (Medium 1),where one can see: (A) formation of sprouts, and (B) elongation ofsprouts.

DEFINITIONS

[0022] As used herein, with no limitation to the scope of the inventionand unless expressly mentioned otherwise, the words below have thefollowing meaning:

[0023] Hypocotyl=small plant stem below cotyledons.

[0024] Cotyledon=embryo leaf which may or may not contain nutritivestocks.

[0025] Leaf=lateral organ representing a laminar expansion of stem.

[0026] Seed=result of the development of ovule after beingfertilization.

[0027] Plantlet=small rooted stem or germinated embryo.

[0028] Callus=mass of non-differentiated cells within higher plantswhich can be formed from cultivated cells or naturally as a reaction toan injury or infection.

[0029] Marker gene=gene which effects are observable over the organism.

[0030] β-Glucuronidase (GUS)=enzyme coding marker gene.

[0031] Col or collect or transition zone=intermediate region between theroot and the stem, with small dimensions. It is an anatomicallyimportant region, since it is where a gradual reorganization ofconducting tissues occurs, when radial (or root) beams becomecollateral, bicollateral or concentric beams.

[0032] t-Student=the t-Student test is a parametric hypothesis test todetermine whether a given result is or is not statistically significant.

[0033] Explant=organ or part of a plant tissue used to start a culture“in vitro”.

[0034] Auxin=class of growth hormones which cause cell elongation,apical dominance, root initiation (rooting), etc. Indol acetic acid(LAA) is a commonly used auxin in tissue culture.

[0035] MS Medium=Murashige & Skoog, “A revised medium for rapid growthand bioassays with tobacco tissue cultures”, Physiol. Plantarum,15:473-497, 1962;

[0036] MSW=MS Macro and Micro-nutrients with vitamins from the medium ofWhite, “A handbook of plant tissue culture”, Lancaster, 1943;

[0037] MS+BAP=as per Machado et al., “Agrobacterium strain specificityand shooty tumor formation in eucalyptus (E. grandis×E. urophylla)”,Plant Cell Rep., 16:299-303, 1997;

[0038] MM=multiplication medium;

[0039] MA=elongation medium;

[0040] ME=rooting medium;

[0041] Medium A, Medium B, Medium C=as per Serrano et al., “Genetictransformation of Eucalyptus globulus through biollistic: complementarydevelopment of procedures for organogenesis form zygotic embryos andstable transformation of corresponding proliferation tissue”, J. Exp.Botany 47 (295), 1996;

[0042] G=as per Gonçalves, “Reversion to juvenility and cloning ofEucalyptus urophylla S. T., Blake in cell in tissue culture systems”.In: Symposium IUFRO em melhoramento genético e produtividade de espéciesflorestais de rápido crescimento, pp.25-30, 1980, Brazil.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The invention refers to a method to insert any gene of interestwithin woody trees, particularly, Eucalyptus spp., by making use ofseeds as a route for transference.

[0044] According to the invention, as a generic view, seeds are used atthe start of germination, when an intense cell division activity occurswithin cotyledonary leaves, thus favoring penetration of bacteriaAgrobacterium. Once developed, the plantlet is cut off and callus areproduced from the cotyledons which, with the addition of antibiotics,herbicides or other compounds to the media of culture, are selected toregenerate transformed plants.

[0045] The method of the invention deals with the generic introductionof a gene, i.e. any gene of interest. Final characteristics of the plantwill depend on the gene being inserted. With no limitation to the scopeof the invention, the following examples can be mentioned:

[0046] a gene granting resistance to a herbicide has as a final result aplant which is tolerant to a given herbicide;

[0047] a gene coding a protease inhibiting protein will generate as afinal product a plant resistant to the attack of given pests.

[0048] A description of the final product can be made depending only onthe characteristics of the gene of interest. Genes changing a phenotypiccharacteristic of interest can be introduced.

[0049] The introduced characteristics may or may not be found in anatural plant. A test of the method of the invention consisted in theintroduction of the gene Lhch1*2 of peas within transgenic tobaccoplants, resulting in an increase in biomass. The confirmation of theefficiency and applicability of the method resulted in an experimentusing woody trees, such as Eucalyptus spp., objects of this application.

[0050] The use of the sonication method with introduction throughAgrobacterium was therefore developed for the genetic transformation ofwoody trees, particularly, Eucalyptus spp., more specifically withindifficult to regenerate species such as E. grandis, E. urophylla andhybrid E. grandis×E. urophylla (HGU), thus obtaining better efficiency.

[0051] The method object of the invention consists of the followingsteps (Eucalyptus spp. is being mentioned for ease of explanation, butit is well understood that this method applies to woody trees ingeneral):

[0052] sterilization of Eucalyptus spp. seeds, e.g. with ethanol andsodium hypochlorite and washing thereof;

[0053] transference of such seeds to an appropriate medium of culture;

[0054] germination for a period between it enters about 2 to 17 days;

[0055] collection of germinated material;

[0056] inoculation with Agrobacterium, particularly Agrobacteriumtumefaciens, containing gene(s) of interest and optionally markergene(s), under concentration of about 10⁷ to 10⁹ cells per ml;

[0057] await inoculation between about 20 to 30 hours in a liquidmedium;

[0058] transference of the material to a solid medium for a periodbetween about 38 and 50 hours in the dark, under temperature betweenabout 25 to 31° C. and ambient humidity;

[0059] transference of the material to start the plantlet growing stepin the light;

[0060] removal of a plantlet leaf obtained between about 5 and 17 daysduring growing step in the light;

[0061] transference of the plantlet leaf to the MS medium containingauxins derived from urea, particularly phenyl urea;

[0062] await germination of the plant tissue for approximately 20 days;

[0063] identification and selection of transformed plantlets;

[0064] keeping the transforming plantlets in a culture and elongationmedium for about 20 days;

[0065] regeneration of the final plant as transformed from one of thefollowing regions: hypocotyl; cotyledon; primary leaves and col.

[0066] Optionally, after the seed germination step, the collectedmaterial can be sonicated for a period of between about 30 and 90seconds, before the inoculation with Agrobacterium.

[0067] The method described in the invention is different from previousmethods for employing seeds, whose essential purpose is to favor genetictransformation. A large number of individuals can therefore be infected,substantially increasing the efficiency of the process.

[0068] Added to this, the invention also encompasses the furtherinclusion of a seed sonication step, leading to an even greater finalregeneration from the hypocotyl, a region that presents a greaterregeneration rate according to the process. Hypocotyl has a regenerationrate higher than 40% and the sonication step raises the rate oftransforming regions, for instance, from the level of 1 to 17% up to 4to 37%.

[0069] The result is a final regeneration rate, which is higher thanthat obtained by previous methods.

[0070] The selection step of regenerated plants is obtained by means ofusing of markers, usual in biotechnological methods, which areintroduced within the Agrobacterium together with the gene of interest,leading to the confirmation of efficiency of this method and allowing adifferentiation in transformed plants, which is an important fact for alarge-scale production process.

[0071] By means of the invention, final transgenic plants as obtainedhave innumerous applications, such as:

[0072] increase in biomass;

[0073] change in lignin percentage;

[0074] stronger protection against pests and diseases;

[0075] stronger resistance to water deficit;

[0076] change in the chemical composition of wood (e.g. lignin,hemicellulose, cellulose, extracts);

[0077] physical change of wood; such as basic density of the wood and/orbark;

[0078] resistance to herbicides;

[0079] use of the plant, in whole or in part, in paper and celluloseindustry, transformation of wood, building and fuel.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0080] Preparation of Means for Tissue Culture

[0081]E. grandis, E. urophylla and hybrid E. grandis×E. urophylla (HGU)seeds were disinfected by making use of the following protocol:

[0082] Wash in ethanol at approximately 70% for about 1 min, wash withsterilized water, transfer to sodium hypochlorite (around 8%volume/volume) and Tween (non-ionic surfactant derived from sorbitanester) (around 0.1% v/v) for about 10 min; after this treatment, thematerial was washed for four times under sterilized water. Seedsgerminated on Petri dishes containing MS culture medium. Table 1presents various culture media as used in different experiments. TABLE 1Culture media used to regenerate different plant tissues: Medium MediumComponents MS MM ME MA 1 2 Macro- MS MS G MS {fraction (1/2 )} MS{fraction (1/2 )} MS nutrients Micro-nutrients MS MS G MS MS MS Na/FeEDTA MS MA 1,5 MS MS MS MS Vitamins MS White G White MS MS Sucrose (%)2.0 2.0 2.0 2.0 2.0 2.0 pH 5.8 5.8 5.8 5.8 5.8 5.8 BAP — 0.2 mg/l — 0.1mg/l — — NAA —  10 μg/l — — 0.465 0.465 mg/l mg/l IBA — — 1.0 mg/l 0.2mg/l — — AGAR (%) 0.6 0.6 0.6 0.6 0.6 0.6 Phenyl urea — — — — 0.5 mg/l1.0 mg/l

Preparation of Inoculation of Agrobacterium tumefaciens Strain LBA 4404

[0083] A colony of this strain was inoculated into liquid Rhizo mediumwith about 100 mg.L⁻¹ of Kanamicin and cultivated under stirring atapproximately 200 rpm for a period of about 48 h, until the grownbacteria reached optical density of about 1-5×10⁸ cells.mL⁻¹. The cellswere centrifuged at approximately 5000 rpm for about 10 min and thepellet was re-suspended in a liquid MS medium containing approximately100 mM of ketoseringone. The bacteria suspension was used for seedinoculation.

Protocol to Transform Seeds by Means of Sonication

[0084] The method described below makes use of the Agrobacterium systemto transform seeds. Added to that method, sonication is applied as apre-treatment of seeds for Agrobacterium infection, according to theworks of Sawahel, “Ultrasound-mediated stable transformation of potatotuber discs”, Biotech. Techniq., 10:821-824, 1996, Trick & Finer 1997,1998 and Santarém et al. 1998. Tissue sonication causes a series ofmicro-injuries on the external epidermis surface and tegument tissues,thus facilitating bacteria penetration and the exposure of embryotissues.

[0085] Seeds were sterilized as previously described, transferred toPetri dishes containing MS medium with approximately 3% sucrose, andkept within controlled growth chambers with photo-periods of about 16 hof light and 8 h in the dark, under temperature of about 26° C. Seedsgerminated within various time intervals (0, 2, 5, 15 and 17 days) weresonicated within periods of about 30 to 90 seconds before beinginoculated with the strain LBA 4404 containing β-glucuronidase (GUS)gene under the control of the 35S promoter of cauliflower mosaic virus(CaMV). Sonication was performed with seeds immersed in about 35 ml ofliquid MS medium in a 100 ml magenta glass flask, using a frequency ofabout 40 Hz. After sonication, seeds were inoculated in a liquid medium(MS) with Agrobacterium tumefaciens (LBA 4404), under density of about10⁷ to 10⁹ cells per ml for a period of about 24 h under constantstirring (approximately 100 rpm). Control seeds (not sonicated) weretransferred immediately after sterilization to the inoculation medium.After the inoculation treatment, seeds were transferred to Petri dishes,containing solid MS medium, for co-cultivation for a period ofapproximately 48 h, at a temperature of about 28° C. in the dark. Seedshave been subsequently washed with sterilized water containing about 200mg.L⁻¹ of cefotaxim, dried over sterilized filter paper and transferredto solid MS medium containing about 100 mg.L⁻¹ of cefotaxim. After atime period of about 7 to 10 days from the complete seed germination,plantlets were evaluated for the expression of β-glucuronidase (GUS)gene. The transformation efficiency considered two parameters:percentage of sprouts with at least a blue point and the transformationgradient. This last parameter represents the average of blue points(transformed areas) from the total of transformed sprouts. Thetransformation gradient indicates the efficiency of transformation.

[0086] Statistical delineation was performed with random blocks with tworepetitions for each treatment. 50 seeds were used per treatment, butnot all seeds germinated. Averages were compared with the t-Student testfor 5% probability. Table 2 presents results of seed transformation bymaking use of the sonication system followed by infection byAgrobacterium. TABLE 2 Effect of sonication on the transformation rateof E. grandis seeds. Figures followed by different letters indicatesignificance (P < 0.05) on the t-Student test. Figures in parenthesisindicate standard average deviation. Control a Sonicated b % Trans- %Days of transformed formation transformed Transformation germinationregions Rate regions rate 0c 2.17 (1.53) 2.0 (1) 4.02 (1.52) 2.0 (1) 2b14.0 (4.0) 4.18 (1.93) 21.72 (0.72) 5.93 (0.33) 5d 1.51 (1.51) 1.5 (1.5)5.54 (2.34) 2.83 (1.83) 15a 17.14 (2.86) 1.12 (0.12) 37.38 (6.61) 2.27(0.27) 17b 11.0 (1.9) 1.12 (0.12) 19.54 (1.89) 4.0 (1)

[0087] Results from this experiment show the potential to transformseeds and plantlets, with the positive sonication effect increasing seedtransformation efficiency. Sonicated seeds with 2 and 15 days ofgermination presented higher transformation rates (21.7 and 37.38%,respectively). There was a differential response from transformedregions, depending on the age of the seed. For seeds germinated for twodays, around 90% of blue segments were located in the cotyledons and inthe col region (between hypocotyl and roots) while, in seeds germinatedfor five days, about 70% of the β-glucuronidase activity was located inthe cotyledons (FIG. 2, A to C). On the other hand, in the plantletswith about 15 to 17 days, about 60% of transformed regions occurred inthe first couple of leaves (FIG. 1, D to F).

Regeneration Systems for Transformed Tissues

[0088] Transformed tissue regeneration is an important step for theprocess of exogenous-to-plant gene transference. Two examples ofregeneration systems, which can be employed to regenerate, transformedtissues such as cotyledons and primary leaves are described below.

[0089] Embodiments that illustrate the invention will now be supplied,not presenting limiting characteristics.

EXAMPLE 1

[0090] Tissue regeneration was obtained with the use of a phenyl urea,by adjusting concentrations for each species of eucalyptus, fromapproximately 0.5 to about 1.0 mg.L⁻¹ (Medium 1 and Medium 2,respectively). Seeds of E. grandis and the hybrid HGU were germinatedfor 20 days under MS medium. Cotyledons, first pair of leaves,hypocotyls and the col of each plantlet were subsequently extracted.Such explants were transferred to media 1 and 2 in equal parts.

[0091] Explants were cultivated for a period of approximately 30 daysunder dark conditions, renewing the medium every about 15 days. Afterthat period, they were transferred to a sprouting induction medium in aculture chamber with a photo-period of about 16 h light/8 h darkness,for about 20 days. The medium used to induce sprouting was Medium 3 (seeTable 1) containing about 0.2 mg.L⁻¹ of phenyl urea. The materialremained in the sprouting-inducing medium for a period of approximately20 days. After that period, it was transferred to a multiplicationmedium (MM) for about 20 days and subsequently to an elongation medium(MA). Evaluations were made based on the quantity of sprouts as formedin both species. In this case, differences in regeneration rates betweenboth genotypes were not considered. For each treatment, 13 explants fromhybrid HGU and 26 explants from E. grandis were used. Results show thatthe formation of sprouts was higher under the dosage of about 0.5 mg.L⁻¹of phenyl urea (Medium 1), 27.3%, while the sprout formation rate forMedium 2 was 16.1%, considering the total quantity of used explants forboth genotypes. Sprout regeneration rate as considered for each tissuefor both genotypes was: cotyledons 16.2%; primary leaves 35.7%;hypocotyls 43.59% and col 2.3%.

EXAMPLE 2

[0092] Based on previous results, plant regeneration was only obtainedfor Medium 1, which presented the highest sprout formation rate. Plantmaterial used was the same. Seeds were sterilized as described in theprevious example, being transferred to Petri dishes containing MS mediumfor about two days, under photo-period of approximately 16 h of lightand temperature of about 26° C. Germinated seeds were sonicated for aperiod of about 30 seconds before being inoculated with LBA 4404 straincontaining β-glucuronidase gene and NPTII gene for resistance againstKanamicin and geneticin G-418. Sonication was performed with seedsimmersed in approximately 35 ml of liquid MS medium in a magenta flask.After sonication, they were inoculated in a liquid medium (MS) withAgrobacterium (see Example 1), for a period of approximately 24 hoursunder constant stirring (aprox.100 rpm). After inoculation treatment,seeds were transferred to Petri dishes containing solid MS medium forco-cultivation for a period of about 48 hours, under temperature ofaround 28° C. in the dark. Seeds were subsequently washed withsterilized water containing about 200 mg.L⁻¹ of cefotaxim, dried oversterilized filter paper and transferred to solid MS medium containingapproximately 100 mg.L⁻¹ of cefotaxim, where they stayed for a period ofabout 15 days, thus allowing for plantlet development.

[0093] From each plantlet, cotyledons were taken off and transferred toMedium 1 with approximately 0.5 mg.L⁻¹ of phenyl urea, about 100 mg.L⁻¹of Kanamicin and about 100 mg.L⁻¹ of cefotaxim, kept in the dark forabout 30 days. The medium was renewed approximately every 15 days.Plantlets were subsequently transferred to the same medium, containingabout 10 mg.L⁻¹ of geneticin G-418 and about 100 mg.L⁻¹ of cefotaximunder light, to induce sprouting, for about 30 days. In that medium, thematerial is subject to strong selection, with survival rate varyingamong used species from about 70 to 90%. Callus and sprouts appearingfrom such treatment were transferred to multiplying medium (MM)containing about 10 mg.L⁻¹ of geneticin G-418, about 100 mg.L⁻¹ ofcefotaxim, for a period of approximately 20 days, when most sproutsdied. Remaining live sprouts were transferred to MA medium until theyreached a length of about 3 to 4 cm. They were subsequently transferredto ME medium for about ten days in the dark and then transferred to agrowing chamber for an approximate period of 20 days. Plantacclimatization took place in a greenhouse, in a mixture of earth andvermiculite.

[0094] Table 3 presents percentage data of sprout formation for E.grandis and the hybrid E. grandis×E. urophylla (HGU). TABLE 3 SPROUTFORMATION PERCENTAGE Plant Structures E. grandis HGU Hypocotyls 25.0 ±8.4 29.2 ± 4.2 Cotyledons 20.8 ± 4.2 24.5 ± 3.7 Leaves 10.4 ± 2.1  8.3 ±4.2

[0095] Other embodiments of the invention will be readily perceived byone skilled in the art from the disclosure included in the presenteddescription or with the practice of the invention as disclosed herein.It is stressed that the examples as presented are particular embodimentsof the invention, whose true scope is expressed by the attached claims.

1. METHOD FOR GENETIC TRANSFORMATION OF WOODY TREES, which comprises thefollowing steps: sterilization and washing of seeds of woody trees;transference of said seeds to an appropriate medium of culture;germination for a period between about 2 and 17 days; collection ofgerminated material; inoculation with Agrobacterium, containing one ormore genes of interest and optionally one or more marker genes, underconcentration between about 10⁷ and 10⁹ cells per ml; await inoculationbetween about 20 and 30 hours in a liquid medium; transference of thematerial to a solid medium for a period between about 38 and 50 hours inthe dark, under temperature between about 25 and 31° C. and ambienthumidity; transference of the material to start plantlet growth in thelight; withdrawal of a plantlet leaf obtained between about 5 and 17days during growth in the light; transference of the plantlet leaf tothe MS medium containing auxins derived from urea; await germination ofthe plant tissue for approximately 20 days; and identification andselection of the transformed plantlets;
 2. METHOD FOR GENETICTRANSFORMATION OF WOODY TREES, which comprises the following steps:sterilization and washing of seeds of woody trees; transference of saidseeds to an appropriate medium of culture; germination for a periodbetween about 2 and 17 days; collection of germinated material;sonication of the material as collected; inoculation with Agrobacterium,containing one or more genes of interest and optionally one or variousmarker genes, under concentration between about 107 and 109 cells perml; await inoculation between about 20 and 30 hours in a liquid medium;transference of the material to a solid medium for a period betweenabout 38 and 50 hours in the dark, under temperature between about 25and 31° C. and ambient humidity; transference of the material to startthe plantlet growth in the light; withdrawal of a plantlet leaf obtainedbetween about 5 and 17 days during growth in the light; transference ofthe plantlet leaf to the MS medium containing auxins derived from urea;await germination of the plant tissue for approximately 20 days; andidentification and selection of transformed plantlets.
 3. METHODaccording to any of claims 1 and 2, characterized by the fact that theauxin derived from urea is phenyl urea.
 4. METHOD according to any ofclaims 1 and 2, characterized by the fact that the gene or genes ofinterest are chosen from a gene granting resistance to herbicides,coding a protease inhibiting protein or changing a phenotypiccharacteristic of interest.
 5. METHOD according to any of claims 1 and2, characterized by the fact that the gene or genes of interest arechosen from NPTII of resistance to Kanamicin and geneticin G-418,Lhcb1*2.
 6. METHOD according to any of claims 1 and 2, characterized bythe fact that the marker gene is β-glucuronidase (GUS).
 7. METHODaccording to any of claims 1 and 2, characterized by the fact that saidmethod results in a final product presenting one or more of thefollowing characteristics: large increase in biomass; change of ligninpercentage; better protection against pests and diseases; betterresistance to water deficit; change in the chemical composition of wood;physical change of wood; such as basic density of the wood and bark, andresistance to herbicides.
 8. METHOD according to any of claims 1 and 2,characterized by the fact that the change in the chemical composition ofwood concerns one or more of the substances chosen from lignin,hemicellulose, cellulose and extracts.
 9. METHOD TO OBTAIN TRANSGENICWOODY TREE PLANTS, which comprises the following steps: sterilizationand washing of seeds of woody trees; transference of said seeds to anappropriate medium of culture; germination for a period between about 2and 17 days; collection of germinated material; inoculation withAgrobacterium, containing one or more genes of interest and optionallyone or more marker genes, under concentration between about 10⁷ and 10⁹cells per ml; await inoculation between about 20 and 30 hours in aliquid medium; transference of the material to a solid medium for aperiod between about 38 and 50 hours in the dark, under temperaturebetween about 25 and 31° C. and ambient humidity; transference of thematerial to start the plantlet growth step in the light; withdrawal of aplantlet leaf obtained between about 5 and 17 days during growth in thelight; transference of plantlet leaf to the MS medium containing auxinsderived from urea; await germination of the plant tissue forapproximately 20 days; identification and selection of transformedplantlets; maintaining transforming plantlets in a multiplication andelongation medium for about 20 days; regeneration of the finaltransformed plant from one of the following regions: hypocotyl;cotyledon; primary leaves and col.
 10. METHOD according to any of theabove claims, characterized by the fact that seed sterilization isperformed with ethanol and sodium hypochlorite.
 11. METHOD according toany of the above claims, characterized by the fact that said seeds areof Eucalyptus spp.
 12. METHOD according to claim 11, characterized bythe fact that the seeds are chosen from Eucalyptus grandis, Eucalyptusurophylla and hybrid Eucalyptus grandis×Eucalyptus urophylla (HGU). 13.METHOD according to any of the previous claims characterized by the factthat said Agrobacterium is Agrobacterium tumefaciens.
 14. PLANT OFTRANSGENIC WOODY TREES, characterized by the fact that it is obtained bymeans of a method according to any of claims 1 to
 13. 15. USE OF THEPLANT OF TRANSGENIC WOODY TREES according to any of claims 1 to 13,characterized by the fact that it produces one or more of the followingcharacteristics: large increase in biomass; change of lignin percentage;better protection against pests and diseases; better resistance to waterdeficit; change in the chemical composition of wood; physical change ofwood; such as basic density of the wood and bark and resistance toherbicides.
 16. USE according to claim 15, characterized by the factthat the change of chemical composition of wood concerns one or more ofthe substances as chosen from lignin, hemicellulose, cellulose andextracts.
 17. USE according to any of claims 15 or 16, characterized bythe fact that it comprises the full or a partial use of the plant in thepaper and cellulose, wood transformation, building or fuel industry.